Permeability flow cell and hydraulic conductance system

ABSTRACT

The present invention relates to devices and methods for measuring the permeability of dentin. More particularly, the invention relates to devices and methods of quickly and accurately measuring the permeability of dentin using a flow cell.

FIELD OF THE INVENTION

The present invention relates to devices and methods for measuring thepermeability of dentin. More particularly, the invention relates todevices and methods of quickly and accurately measuring the permeabilityof dentin using a flow cell.

BACKGROUND OF THE INVENTION

Tooth sensitivity affects numerous people. It is often caused by eatingor drinking something hot, cold, sweet or acidic. Under normalconditions, the papal chamber, which houses the blood vessels andnerves, is surrounded by the dentin, which in turn is covered by theenamel in the tooth crown, and the gums that surround the tooth. Overtime, the enamel covering can get thinner, thus providing lessprotection. The gums can also recede over time, exposing the underlyingroot surface dentin.

The dentin contains a large numbers of pores or tubule orifices that runfrom the outside of the tooth to the nerve at its center. When thedentin is exposed, these tubule orifices can be stimulated bytemperature changes or certain foods. The hydrodynamic theory of dentinsensitivity states that stimuli applied to exposed dentin tubuleorifices cause a movement of fluids in the tubules which, in turn,stimulates nerves in the pulp.

The well-know Pashley method for determining dentin permeability hasbeen used as an in vitro model for screening agents which have been usedto desensitize dentin. In this method, fluid is forced from an inletacross (or through) one side of a dentin disc sample to the other sideand, then, its flow rate measured to determine the rate of fluid flowacross the dentin sample. The prepared disc sample of dentin is securedin a split-chamber device, clamped between two paired “O” rings.

Certain limitations, however, exist regarding the Pashley method andgenerally relate to inherent inaccuracies affecting the overall accuracyof the method's permeability measurements. Moreover, the design of theflow cell used in the Pashley method does not allow for easy removal ofthe dentin sample during dentin permeability analysis to, for exampleperform further challenge to the dentin sample's surface and, then,return the dentin sample back to the flow cell for continued analysis.

Another limitation of the Pashley method relates to its inability tostandardize the flow rates obtained across different dentin samples whenpermeability data for more than one dentin sample is necessary ordesired.

As a result of these limitations, large sample sizes are required toachieve statistically significant dentin permeability readings.

The search for faster and more accurate methods of measuring thepermeability of dentin using a modified flow cells and/or permeabilitymeasurement methods continues. Desired aspects for these methods includehigh accuracy and throughput (i.e., performing technology testingquickly and reliably producing sound data), data separation, errorreduction, robustness, repeatability, and use with additional testingmethods.

SUMMARY OF THE INVENTION

The present invention relates to devices, apparatus and methods formeasuring the permeability of dentin.

In one embodiment, the present invention relates to a flow cell formeasuring hydraulic conductance of dentin samples, comprising:

-   -   a. a flow inlet channel;    -   b. a flow outlet channel in flow communication with the flow        inlet channel;    -   c. at least one dentin sample securing mechanism positioned        between the flow inlet channel and the flow outlet channel for        securing a dentin sample; and    -   d. at least one venting channel having an inner opening from        which the venting channel extends outwardly from the flow cell,        the venting channel positioned for receiving any air in the form        of at least one air bubble that might accumulate under a dentin        sample secured by the securing mechanism after introduction of a        fluid into the flow cell through the flow inlet channel,        wherein the venting channel forms a positive angle θ relative to        a bottom side of a horizontal cross-sectional plane through the        bottom component, which horizontal cross-sectional plane        intersects the inner opening of the venting channel such that        the angle θ ranges from greater than about 0° to less than about        90°, the angle θ being measured counter clockwise from the        bottom side of the horizontal cross-sectional plane and having        its vertex at the point of intersection of the inner opening and        the horizontal cross-sectional plane.

In another embodiment, the present invention relates to a flow cell formeasuring hydraulic conductance of a dentin sample, comprising:

-   -   A. a bottom component comprising:        -   i. an inner chamber;        -   ii. at least one flow inlet channel in flow communication            with the inner chamber;        -   iii. at least one flow outlet channel in flow communication            with the flow inlet channel and the inner chamber;        -   iv. at least one venting channel in flow communication with            the inner chamber and having an inner opening where the            venting channel joins with the inner chamber; and        -   v. an opening at the top of the bottom component for            accessing the inner chamber,    -   B. a removable lid for covering the opening of the bottom        component, the lid having a flow outlet channel positioned for        receiving and permitting the outflow of fluid diffusing through        (or across) the dentin sample from the flow inlet channel; and    -   C. at least one washer adjacent to the lid and/or to the bottom        component for securing the dentin sample within the flow cell,        wherein the venting channel forms a positive angle θ relative to        a bottom side of a horizontal cross-sectional plane through the        bottom component, which horizontal cross-sectional plane        intersects the inner opening of the venting channel such that        the angle θ ranges from greater than about 0° to less than about        90°, the angle θ being measured counter clockwise from the        bottom side of the horizontal cross-sectional plane and having        its vertex at the point of intersection of the inner opening and        the horizontal cross-sectional plane.

In a further embodiment, the present invention relates to an apparatusfor measuring hydraulic conductance of dentin samples, comprising;

-   -   A. a flow cell comprising:        -   a. a bottom component comprising:            -   i. an inner chamber;            -   ii. at least one flow inlet channel in flow                communication with the inner chamber;            -   iii. at least one venting channel in flow communication                with the inner chamber and having an inner opening where                the venting channel joins with the inner chamber wherein                the venting channel forms a positive angle θ relative to                a bottom side of a horizontal cross-sectional plane                through the bottom component, which horizontal                cross-sectional plane intersects the inner opening of                the venting channel such that the angle θ ranges from                greater than about 0° to less than about 90°, the angle                θ being measured counter clockwise from the bottom side                of the horizontal cross-sectional plane and having its                vertex at the point of intersection of the inner opening                and the horizontal cross-sectional plane; and            -   iv. an opening at the top of the bottom component for                accessing the bottom component;        -   b. a removable lid for covering the opening of the bottom            component, the lid having a flow outlet channel positioned            for receiving and permitting the outflow of fluid diffusing            through the dentin sample from the flow inlet channel; and        -   c. at least one washer adjacent to the lid and/or to the            bottom component for securing the dentin sample within the            flow cell.    -   B. a pumping mechanism for pumping a fluid into the flow cell        through the flow inlet channel, through the dentin sample and        out of the flow cell through the flow outlet channel; and    -   C. at least one measuring device suitable for measuring and/or        determining hydraulic conductance through a dentin sample.

Yet another embodiment of the present invention relates to a method formeasuring the hydraulic conductance through a dentin sample, comprisingthe steps of:

-   -   A. providing a flow cell for measuring hydraulic conductance of        dentin samples comprising:        -   a. a bottom component comprising:            -   i. an inner chamber;            -   ii. at least one flow inlet channel in flow                communication with the inner chamber;            -   iii. at least one venting channel in flow communication                with the inner chamber and having an inner opening where                the venting channel joins with the inner chamber wherein                the venting channel forms a positive angle θ relative to                a bottom side of a horizontal cross-sectional plane                through the bottom component, which horizontal                cross-sectional plane intersects the inner opening of                the venting channel such that the angle θ, ranges from                greater than about 0° to less than about 90°, the angle                θ being measured counter clockwise from the bottom side                of the horizontal cross-sectional plane and having its                vertex at the point of intersection of the inner opening                and the horizontal cross-sectional plane; and            -   iv. an opening at the top of the bottom component for                accessing the base portion,        -   b. a removable lid for covering the opening of the bottom            component, the lid having a flow outlet channel positioned            for receiving and permitting the outflow of fluid diffusing            through the dentin sample from the flow inlet channel; and        -   c. at least one washer adjacent to the lid and/or to the            bottom component for securing the dentin sample within the            flow cell;    -   B. placing the dentin sample adjacent at least one washer;    -   C. sealing the flow cell with the removable lid;    -   D. providing a pumping mechanism for pumping a fluid into the        flow cell through the flow inlet channel;    -   E. introducing a fluid into the flow cell such that the fluid        fills the inner chamber and contacts the dentin sample;    -   F. tilting the flow cell such that a negative angle φ is formed        relative to a top side of a horizontal cross-sectional plane        through the bottom component to remove any accumulated air in        the form of at least one air bubble generated after introduction        of the fluid into the flow cell, which horizontal        cross-sectional plane intersects the inner opening of the        venting channel such that the angle φ ranges from greater than        about 0°, the angle φ being measured clockwise from the top side        of the horizontal cross-sectional plane and having its vertex at        the point of intersection of the inner opening and the        horizontal cross-sectional plane;    -   G. pumping the fluid into the flow cell through the flow inlet        channel such that the fluid diffuses through the dentin sample        and the flow outlet channel; and    -   H. measuring the flow rate of the fluid pumped into the flow        cell to determine the hydraulic conductance through dentin        sample.

In another embodiment, the present invention relates to a flow cell,comprising:

-   -   i. a flow inlet channel having an inner opening, the flow inlet        extending from the inner opening outwardly from the flow cell;    -   ii. a flow outlet channel in flow communication with the flow        inlet channel; and    -   iii. at least one reversible dentin sample securing mechanism        positioned to secure a dentin sample between the flow inlet        channel and the flow outlet channel, the dentin sample securing        mechanism comprising a securing mechanism and at least one        washer positioned adjacent the securing mechanism for receiving        or contacting a dentin sample, the washer having at least one        flat side for (optionally, leak free or substantially leak free)        contact with a dentin sample.

In yet another embodiment, the present invention relates to a flow cell,comprising:

-   -   a. a bottom component comprising:        -   i. an inner chamber;        -   ii. at least one flow inlet channel in flow communication            with the inner chamber;        -   iii. at least one flow outlet channel in flow communication            with the flow inlet channel and the inner chamber; and        -   iv. an opening at the top of the bottom component for            accessing the inner chamber,    -   b. a removable lid for covering the opening of the bottom        component, the lid having a flow outlet positioned for receiving        and permitting the outflow of fluid diffusing through the dentin        sample from the flow inlet channel; and    -   c. at least one washer having at least one flat side for        contacting a dentin sample or optionally at least one pair of        flat sides, one flat side opposite (or substantially opposite)        the other flat side, wherein one flat side of the washer        contacts the lid and/or the bottom component and the other side        of the pair of flat sides is positioned to contact the dentin        sample for securing the dentin sample within the flow cell.

Another embodiment of the present invention relates to an apparatus formeasuring hydraulic conductance of dentin samples, comprising;

-   -   a. a flow cell, comprising:        -   i. a flow inlet channel;        -   ii. a flow outlet channel in flow communication with the            flow inlet channel; and        -   iii. at least one reversible dentin sample securing            mechanism positioned to secure a dentin sample between the            flow inlet channel and the flow outlet channel, the dentin            sample securing mechanism comprising a securing mechanism            and at least one washer having at least one flat side for            contacting a dentin sample or optionally at least one pair            of flat sides, one flat side opposite (or substantially            opposite) the other flat side, wherein one flat side of the            pair of flat sides contacts the dentin sample and the other            flat side of the pair of flat sides contacts the securing            mechanism;    -   b. a pumping mechanism for pumping a fluid into the flow cell        through the flow inlet channel, through the dentin sample and        out of the flow cell through the flow outlet channel; and    -   c. at least one flow meter for measuring the flow of fluid        pumped into the flow cell and through the dentin sample.

In a further embodiment, the present invention relates to an apparatusfor measuring hydraulic conductance of dentin samples, comprising;

-   -   a. a flow cell comprising;        -   i. a flow inlet channel having an inner opening, the flow            inlet extending from the inner opening outwardly from the            flow cell:        -   ii. a flow outlet channel in flow communication with the            flow inlet channel; and        -   iii. at least one dentin sample securing mechanism            positioned to secure a dentin sample between the flow inlet            channel and the flow outlet channel;    -   b. a pumping mechanism for exerting a pressure to pump a fluid        into the flow cell through the flow inlet channel, through a        dentin sample and out of the flow cell through the flow outlet        channel;    -   c. a pressure regulator in flow communication with the pumping        mechanism for regulating the pressure exerted by the pumping        mechanism; and    -   d. at least one flow meter in measuring contact with a fluid        pumped by the pumping mechanism for directly measuring the flow        rate of a fluid pumped into the flow cell and through the dentin        sample.

A further embodiment of the present invention relates to an apparatusfor measuring hydraulic conductance of dentin samples, comprising afluid flow rate standardization mechanism, comprising:

-   -   i. a pumping mechanism for pumping a fluid through the        apparatus;    -   ii. at least one adjustable high precision flow regulator for        maintaining a pressure in the apparatus of less than or equal to        5 psi without fluctuations in the maintained pressure of greater        than or equal to ±about 0.1 psi for a period of at least 10        minutes; and    -   iii. at least one fluid flow meter for measuring the fluid flow        rate across a dentin sample,        wherein the fluid flow rate is standardized across different        dentin samples for establishing a single fluid flow rate as the        control against which the flow rate of the different dentin        samples, after modification of the dentin samples, is compared.

Another embodiment of the present invention relates to a method formeasuring the hydraulic conductance through a dentin sample, comprisingthe steps of:

-   -   a. providing a pumping mechanism for pumping a fluid through an        apparatus;    -   b. pumping a fluid through the apparatus    -   c. providing at least one adjustable high precision flow        regulator for maintaining a pressure in the apparatus of less        than or equal to 20 psi without fluctuations in the maintained        pressure, for a period of at least 10 minutes, of greater than        or equal to ±about 0.1 psi;    -   d. at least one fluid flow meter for measuring the fluid flow        rate across a dentin sample;    -   e. directing the fluid flow through a dentin sample; and    -   f. noting the fluid flow rate through the dentin sample        indicated by the fluid flow meter wherein steps a. through f.        are repeated for at least one other dentin sample and further        wherein the flow regulator is adjusted such that the flow rate        across the at least one other dentin sample(s) equals the flow        rate of the first dentin sample.

Another embodiment of the present invention relates to an apparatus formeasuring hydraulic conductance of dentin samples, comprising;

-   -   a. a flow cell comprising:        -   i. a flow inlet channel;        -   ii. a flow outlet channel in flow communication with the            flow inlet channel; and        -   iii. at least one dentin sample securing mechanism            positioned between the flow inlet channel and the flow            outlet channel;    -   b. a pumping mechanism for pumping a fluid into the flow cell        through the flow inlet channel, through a dentin sample and out        of the flow cell through the flow outlet channel;    -   c. a first flow rate meter in measuring contact with a fluid        pumped by the pumping mechanism and calibrated to measure fluid        flowing at a flow rate range of from about 0 microliters per        minute to about 200 microliters per minute; and    -   d. a second flow rate meter for measuring the flow rate of the        fluid pumped by the pumping mechanism to confirm that a fluid        pumped by the pumping mechanism is flowing at a rate within the        flow rate calibration range of from about 0 microliters per        minute to about 200 microliters per minute.

In a still further embodiment, the present invention relates to a methodfor measuring the hydraulic conductance through a dentin sample,comprising the steps of:

-   -   A. providing a flow cell for measuring hydraulic conductance of        dentin samples comprising:        -   a. a bottom component comprising:            -   i. an inner chamber;            -   ii. at least one flow inlet channel in flow                communication with the inner chamber;            -   iii. at least one venting channel in flow communication                with the inner chamber;            -   iv. an opening at the top of the bottom component for                accessing the bottom component,        -   b. a removable lid for covering the opening of the bottom            component, the lid having a flow outlet channel positioned            for receiving and permitting the outflow of fluid diffusing            through the dentin sample from the flow inlet channel; and        -   c. at least one washer adjacent to the lid and/or to the            base for securing a dentin sample within the flow cell;    -   B. placing the dentin sample adjacent the washer of the flow        cell;    -   C. providing a mechanism for pumping a fluid into the flow cell        through the flow inlet channel;    -   D. pumping a fluid into the flow cell through the flow inlet        such that the fluid diffuses through the dentin sample and the        flow outlet channel;    -   E. providing a first flow rate meter in measuring contact with        the fluid and calibrated to measure fluid flowing at a flow rate        range of from about 0 microliters per minute to about 200        microliters per minute;    -   F. measuring the flow rate of the fluid pumped into the flow        cell using the first flow rate meter to determine the hydraulic        conductance through dentin sample;    -   G. providing a second flow rate meter;    -   H. measuring the flow rate of the fluid to confirm that the        fluid is flowing at a rate within the flow rate calibration        range; and    -   I. determining the hydraulic conductance through the dentin        sample.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, directed to one of ordinary skill in the art, is setforth in the specification, which makes reference to the appendeddrawings, in which:

FIG. 1 is a vertical sectional view of the prior art flow cell for usein measuring the permeability of dentin with a dentin sample in placeprior to the sealing of the cell;

FIG. 2 is a vertical sectional view of the flow cell of FIG. 1 with adentin sample in place after the sealing of the flow cell;

FIG. 3 is a top view of the bottom component of the flow cell for use inthe present invention;

FIG. 4 is a vertical sectional view of FIG. 3 along the 4-4 plane;

FIG. 5 is a top view of the top component of the flow cell for use inthe present invention;

FIG. 6 is a vertical sectional view of FIG. 5 along the 6-6 plane;

FIG. 7 is a vertical sectional view of the flow cell for use in thepresent invention with a dentin sample in place prior to the sealing ofthe cell;

FIG. 8 is a vertical sectional view of the flow cell for use in thepresent invention with a dentin sample in place after the sealing of thecell;

FIG. 9 shows representative embodiments a to g of washers useful in thepresent invention;

FIG. 10 is a flow cell positioned (e.g., as by rotation or tilting) topermit venting of air bubbles from the flow cell; and

FIG. 11 is a schematic drawing of the equipment or system lay-out forthe method of measuring the permeability of dentin according to thecurrent invention.

DETAILED DESCRIPTION OF THE INVENTION

The devices, apparatus and methods of the present invention cancomprise, consist of, or consist essentially of the essential elementsand limitations of the invention described herein, as well any of theadditional or optional components, or limitations described herein.

The term “comprising” (and its grammatical variations) as used herein isused in the inclusive sense of “having” or “including” and not in theexclusive sense of “consisting only of.” The terms “a” and “the” as usedherein are understood to encompass the plural as well as the singular.

All patent documents incorporated herein by reference in their entiretyare only incorporated herein to the extent that they are notinconsistent with this specification.

The term “flat” as used herein means having a horizontal surface withouta slope, tilt, or curvature; or, having a smooth, even, level surface.

As used herein the phrase “reversible securing mechanism” means asecuring mechanism which does not secure items (such as a dentin sample)permanently (i.e., as by gluing or cementing), but which after the itemis secured, permits adjustment such that the item can be readilyreturned to its unsecured state. The present invention is a devices andmethods for measuring the permeability of dentin.

FIG. 1 is a vertical sectional view of the prior art flow cell for usein measuring the permeability of dentin prior to sealing the flow cell.The prior art flow cell is generally cylindrical in shape. The figureshows the two-part cell with base component 10 and lid component 50. Lidcomponent 50 includes inner surface 52, outer surface 54, screw threads58 disposed on inner surface 52, and through-hole 56.

Base component 10 includes inner surface 12, outer surface 14, lip 16,screw threads 18 disposed on outer surface 14, and inlet 22 and outlet24 channels. Inlet 22 and outlet 24 channels have “press-fit”connections to inlet and outlet tubes. As used herein, the term “pressfit” (also referred to as “interference fit” or “friction fit”) meansthe fastening of two parts achieved by friction between the parts afterthe parts are joined (e.g., as by pressing or pushing) together, insteadof by any other type of fastening. The cylindrical shape of basecomponent 10 defines inner chamber 20. The components which occupy innerchamber 20 of base component 10 for use in measuring the permeability ofdentin sample 70 include top spacer 32 and bottom spacer 36, as well as“O”-rings 42 and 46 and larger sized “O”-rings 44, and 48. Top spacer 32has through-hole 34, and bottom spacer 36 has through-hole 38.

The components which occupy inner chamber 20 of base component 10 of theprior art flow cell are assembled in the form of a stack as follows:bottom spacer 36 is placed on “O”-ring 48, which “O” ring 48 rests onthe inner surface 12 of base component 10. “O”-rings 46 and 44 areplaced on bottom spacer 36. Second side 74 of dentin sample 70 is placedon “O”-ring 46. “O”-ring 42 is placed on first side 72 of dentin sample70. Top spacer 32 is placed on “O”-rings 42 and 44.

FIG. 2 shows the prior art flow cell after the sealing of the cell. Toseal the cell, lid component 50 is screwed onto base component 10, withscrew threads 58 disposed on inner surface 52 of lid component 50matched with screw threads 18 disposed on outer surface 14, of basecomponent 10.

The permeability of dentin sample 70 is measured using the prior artflow method and cell in the following manner. Once the two-part cell isassembled, outlet channel 24 is sealed. Pressure is used to induce fluid(e.g., distilled water) flow in inlet channel 22. Fluid flows into theportion of inner chamber 20 of base component 10 below dentin sample 70from inlet channel 22. As the fluid pressure rises in the portion ofinner chamber 20 below dentin sample 70, the fluid flows throughthrough-hole 38 of bottom spacer 36. Increased fluid pressure theninitiates fluid flow through (or across) the dentin sample 70 (i.e.,through or across the dentin tubule or orifices in the dentin sample).Fluid flow continues through through-hole 34 of top spacer 32, and exitsprior art flow cell through through-hole 56 of lid component 50.

The limitations of the Pashley method involve inherent inaccuracies inthe Pashley flow cell. These inaccuracies include stacking error due tothe number of flow cell components (i.e., the “O”-rings [four] andspacers [two]) as well as increased leaking potential. Leaks around thedentin sample are often caused by the user inaccurately placing the“O”-rings and spacers when assembling the cell. These leaks around thedentin sample result in an inaccurate measurement of permeabilitythrough the dentin.

Moreover, the “O”-rings used with the Pashley flow cell have roundcross-sections. These “O”-rings have a single line of contact with thedentin sample once the components are arranged and the flow cell sealedas illustrated in FIG. 2. If the “O”-rings are formed of a “stiff”material, the risk of leakage in the system is increases. To alleviatethis risk, the user typically adds extra vertical pressure to the“O”-rings when sealing the flow cell. Such additional vertical pressure,however, creates (or increases) the risk of damaging the dentin sample.On the other hand, if the “O”-rings are formed of a “soft” material, the“O”-rings will deform, and flatten against the dentin sample, changingthe area of the dentin sample exposed to the fluid in the flow cell.Such inconsistency in the area of the dentin sample exposed to the fluidin the flow cell can result in inconsistent measurements of permeabilitythrough the dentin.

Another issue with the Pashley flow cell is that during the assembly ofthe cell, lid component 50 is screwed onto base component 10, with screwthreads 58 disposed on inner surface 52 of lid component 50 matched withscrew threads 18 disposed on outer surface 14, of base component 10,rotating lid component 50 relative to base component 10. The rotationalmovement of the lid component 50 with respect to the base component 10often causes rotation of the dentin sample, the “O”-rings and/or thespacers, which can result in leaks (or increased leakage) around thedentin sample. These leaks around the dentin sample result in aninaccurate measurement of permeability through the dentin.

Yet another issue with the Pashley flow cell relates to the previouslymentioned “press-fit” connections between inlet and outlet tubes and theinlet channel 22 and outlet 24 channel, respectively. Such “press-fit”connections often leak, especially under pressure, resulting ininaccuracies in flow rate measurements.

Furthermore, the Pashley method fails to provide for standardization offlow rates across a variety of different dentin samples whenpermeability data of more than one dentin sample is necessary ordesired. Such standardizing of flow rates across different dentinsamples would alleviate the need, in comparative analyses, to correctfor such dentin sample variables as thickness and porosity. Typicaldentin permeability flow rate methods measure the fluid flow rate acrossa particular dentin sample at a set generated (and maintained) pressure.For example, at a set pressure of, say, 0.75 psi, the fluid flow rate ofone dentin sample might read 3 ul/min, but the fluid flow rate of asecond dentin sample at the same (0.75 psi) pressure might read 10ul/min, and the fluid flow rate of a third dentin sample at the same(0.75 psi) pressure might read 1 ul/min, etc. Differences in thementioned dentin thickness and porosity variables primarily account forthese fluid flow rate differences at a set (and maintained) pressure. Tocorrect for these difference in fluid flow rates, the fluid flow ratesof the different dentin samples are typically normalized. The term“normalized” as used herein means dividing every measurement for a givendentin sample by that sample's baseline (i.e., prior to treatment) flowrate measurement (see “Residual Permeability” formula at Example 1). Byregulating the system pressures to less than or equal to 30 psi (orabout 30 psi) and maintaining the system pressure with minimalfluctuation (i.e., less than or equal to ±0.1 psi) using a highprecision pressure regulator, the present invention permits suchstandardization (i.e., establishing the same fluid flow rate for eachdentin sample) across a variety of different dentin samples.

FIGS. 3 through 8 are views of the two-part flow cell 100 for use in thepresent invention. FIG. 3 is a top view of the bottom component 110 offlow cell 100, while FIG. 4 is a vertical sectional view of FIG. 3 alongthe 4-4 plane. Bottom component 110 of the flow cell includes bottomsurface 112, top surface 114, first indent 116, groove 118, secondindent 119 which defines inner chamber 130, fastener blind holes 135 (orother suitable mechanism for engaging fasteners), inlet channel 144 inflow communication with optional secondary inlet channel 142, andventing channel 148 in flow communication with optional secondaryventing channel 146. In certain embodiments, inlet channel 144 andventing channel 148 are positioned opposite or substantially oppositeeach other. Inlet channel 144 and venting channel 148 each have an innerend, 144 a and 148 a, respectively, the inner openings, 144 a and 148 a,joining inlet channel 144 and venting channel 148 to the inner chamber130. The inner openings, 144 a and 148 a, further define the point fromwhich inlet channel 144 and venting channel 148 extend outwardly fromthe flow cell 100. The bottom component 110 comprises an opening 132 atthe top of the inner chamber 130 for accessing the inner chamber 130.Inlet channel 144 is positioned in flow communication with inner chamber130. Venting channel 148 is also positioned in flow communication withinner chamber 130. Inlet channel 144 and venting channel 148 (or, ifpresent, optional secondary inlet channel 142 and optional secondaryventing channel 146) can be, optionally, threaded to receive thecompatibly threaded ends of inlet and outlet tubes 238 and 254,respectively. Optionally, and as shown on the vertical cross-sectionalview of the bottom compartment at FIG. 4, venting channel 148 forms apositive angle θ relative to a bottom side of a horizontalcross-sectional plane XY through the bottom component 110 andintersecting the inner end 148 a of venting channel 148, angle θ havingits vertex at the point of intersection of the inner end 148 a and thehorizontal cross-sectional plane XY. Angle θ, as measured from thebottom side of the horizontal cross-sectional plane XY counter clockwise(as illustrated at FIG. 4), ranges from greater than about 0° to lessthan about 90°, optionally from about 15 to about 75, optionally fromabout 35 to about 55, or, optionally about 60°. Optionally, and as shownon the vertical cross-sectional view of the bottom compartment at FIG.4, inlet channel 144 forms a positive angle φ relative to a bottom sideof a horizontal cross-sectional plane X′Y′ through the bottom component110 and intersecting the inner end 144 a of inlet channel 144, angle φhaving its vertex at the point of intersection of the inner end 144 aand the horizontal cross-sectional plane X′Y′. Angle φ, as measured frombottom side of the horizontal cross-sectional plane X′Y′ counterclockwise (as illustrated at FIG. 4), ranges from about 0° to less thanor equal to about 270°, optionally from about 90° to about 180°,optionally from about 100° to about 130°, or, optionally about 116°.(For purposes of illustrating angles θ and φ, FIG. 4 shows thecross-sectional planes XY and X′Y′ perpendicular to, and coming out ofthe paper along the x axis and the x′ axis, respectively.)

FIG. 5 is a top view of the lid 150 for flow cell 100, while FIG. 6 is avertical sectional view of FIG. 5 along the 6-6 plane. The lid 150 ofthe flow cell includes bottom surface 152, top surface 154, bottomgroove 158, optional fastener through-holes 175 (or other suitablemechanism for engaging fasteners), and flow outlet channel 160. Flowoutlet channel 160 is defined by walls 156 on lid 150 vertically,radially and conically skewed toward the center of the lid 150,increases the diameter of the out flow of fluid through flow outletchannel 160.

In certain embodiments, lid 150 and bottom component 110 are shaped tofit one in the other so as to permit a secure engagement between the twocomponents. The lid 150 and bottom component 110 components may beformed from machined glasses; woods; metals, such as stainless steel;plastics, such as polymethyl methacrylate (PMMA) or polycarbonate (PC);or a combination of these materials. In one embodiment, lid 150 andbottom component 110 are formed from (e.g., by machining) opticallyclear or transparent PMMA, such as that available from MacMaster-Carr(Catalogue #8560K912 or #8560K265) of Robbinsville, N.J. The advantageof using a clear (e.g., optically clear or transparent) material informing flow cell 100 is that clear materials allow “line of sight” intothe cell or otherwise makes the contents of the cell visible to theunaided eye to, for example, help in visually determining whether allair in the form of air bubble(s) has been purged from the portion ofinner chamber 130 below dentin sample 190 flows. An air bubble belowdentin sample 190 decreases the area of dentin sample 190 through whichfluid can flow through. As a reminder, the inability to consistentlydetermine the area of the dentin sample exposed to the fluid flow cell100 may result in an inconsistent measurement of permeability throughthe dentin.

FIG. 7 shows the flow cell for use in the present invention with dentinsample 190 in place prior to the sealing of the flow cell, while FIG. 8shows the flow cell with a dentin sample in place after the sealing ofthe cell. The components which occupy the flow cell of the presentinvention include first and second washers 182 and 184, and dentinsample 190. Dentin sample 190 has first side 192 and second side 194.

The flow cell is assembled as follows. First washer 182 is placed ingroove 118 of bottom component 110. Second washer 184 is placed inbottom groove 158 of lid 150. In certain embodiments, groove 118 ofbottom component 110, and bottom groove 158 of lid 150 are machined tofit the width dimensions of any washer(s) used (such as washers 182 and184) so as to reduce, minimize or prevent any displacement of thewasher(s): i) as the components of the flow cell are being secured foruse (e.g., testing and/or fluid flow measurement); and/or ii) duringactual use (e.g., testing and/or fluid flow measurement). In otherembodiments, groove 118 can be additionally machined so as to avoidobstructing or otherwise interfering with fluid and/or air bubble flowinto and/or through venting channel 148. Second side 194 of dentinsample 190 is placed on first washer 182. Washer 184 is placed on firstside 192 of dentin sample 190. To complete sealing of the cell, lid 150is fastened onto bottom component 110, using fasteners 186. In theexemplified embodiment, the fasteners 186 are screws which pass throughoptional fastener through-holes 175 of lid 150 and are anchored in/byfastener blind holes 135 of bottom component 110 having screw holessuitable for engaging the screws so that the screws adjustably tightenand seal the lid 150 on to bottom component 110. The flow cell,including lid 150 and bottom component 110 is referred to as flow cell100. Fasteners 186 may be formed of materials such as stainless steel.Fastener through-holes 175 and fastener blind holes 135 are machined tofit and engage fasteners 186.

Alternatively, the assembly of lid 150 on to bottom component 110 can beaccomplished by the use of other adjustable fastening mechanisms, suchas nails, dowels, clamps, straps, bolts (e.g., screw-type), or any otherfastening mechanism suitable for providing a leak proof (orsubstantially leak proof) seal and allow for ready disassembly andassembly. Optionally, the fastening mechanism can operate by friction orinterference fit so long as the friction or interference fit canwithstand the fluid pressures necessary for practicing the presentinvention.

The “washers” 182 and 184 of the present invention are have at least oneflat side for contacting a dentin sample, optionally the washers aresquare washers or washers having at least one pair of flat sides, oneflat side opposite (or substantially opposite) the other flat side ofthe pair, such that one flat side of the pair contacts the lid 150and/or the base and the other flat side of the pair is positioned tocontact a dentin sample for securing the dentin sample within the flowcell as illustrated in FIG. 8. In one embodiment, washers 182 and 184 ofthe present invention are “O” rings with square cross-sections as shownin FIG. 9 a. First side (182′ and 184′) and second side (182″ and 184″)of washers 182 and 184 are flat.

Embodiments of washers useful in the present invention include, but arenot limited to, such examples as shown in FIGS. 9 a to 9 g. FIG. 9 ashows washer cross-sections 182 a and 184 a of a rectangular “O” ring.First side (182 a′ and 184 a′) and second side (182 a″ and 184 a″) ofwasher cross-sections 182 a and 184 a are flat. Washer cross-sections182 b and 184 b of hexagonal “O” ring are shown in FIG. 9 b. First side(182 b′ and 184 b′) and second side (182 b″ and 184 b″) of washercross-sections 182 b and 184 b are flat. In FIG. 9 c, washercross-sections 182 c and 184 c of trapezoidal “O” ring. First side (182c′ and 184 c′) and second side (182 c″ and 184 c″) of washercross-sections 182 c and 184 c are flat. Washer cross-sections 182 d and184 d of rounded rectangular “O” ring are shown in FIG. 9 d. First side(182 d′ and 184 d′) and second side (182 d″ and 184 d″) of washercross-sections 182 d and 184 d are flat. In FIG. 9 e, washercross-sections 182 e and 184 e of race-track shaped “O” ring. First side(182 e′ and 184 e′) and second side (182 e″ and 184 e″) of washercross-sections 182 e and 184 e are flat. FIG. 9 f shows washercross-sections 182 f and 184 f of a single flat-sided “O” ring variant.Sides (182 f″ and 184 f″) of washer cross-sections 182 f and 184 f areflat. FIG. 9 g shows washer cross-sections 182 g and 184 g of a singleflat-sided “O” ring variant. Sides (182 g″ and 184 g″) of washercross-sections 182 g and 184 g are flat. It should be understood thatthe shape of the cross-sections of washers 182 and 184 need not be thesame, but may be independently different in shape such that washer 182may have, for example, the cross-sectional shape illustrated at FIG. 9 cand washer 184 may have the cross-sectional shape illustrated at FIG. 9g.

Washers 182 and 184 may be made of, silicon, rubber or soft plastic.Examples of such silicon, rubber or soft plastic materials, include, butare not limited to, butadiene rubber, butyl rubber, chlorosulfonatedpolyethylene, epichiorohydrin rubber, ethylene propylene diene monomer,ethylene propylene rubber, fluoroelastomer, nitrile rubber,perfluoroelastomer, polyacrylate rubber, polychloroprene, polyisoprene,polysulfide rubber, sanifluor, silicone rubber and styrene butadienerubber) and thermoplastics (including, but not limited to, thermoplasticelastomer; thermoplastic polyolefin, thermoplastic polyurethane,thermoplastic etheresterelastomers, thermoplastic polyamide(s), meltprocessible rubber thermoplastic vulcanizate) and mixtures thereof. Inone embodiment, the washers may be rubber “O”-rings supplied byMcMaster-Carr (Catalogue #4061T114) of Robbinsville, N.J.

The permeability of dentin sample 190 is measured using flow cell 100 inthe present invention in the following manner. Once the two-part flowcell 100 is assembled, pressure is used to initiate and maintain fluid(e.g., distilled water) flow in inlet channel 144, optionally via 1secondary inlet channel 142. In the case FIG. 8, fluid flows fromoptional secondary inlet channel 142 into inlet channel 144, and intothe portion of inner chamber 130 of bottom component 110 below dentinsample 190. Initially, venting channel 148 (and optional secondaryventing channel 146), are kept open so that residual air in the form ofair bubble(s) located in the portion of inner chamber 130 below dentinsample 190 flows into venting channel 148 and exits flow cell 100 (insome embodiments, through optional secondary channel 146). When theresidual air has been removed, venting channel 148 (and/or optionalsecondary venting channel 146) is closed. When venting channel 148(and/or optional secondary venting channel 146) is closed, fluidpressure rises in the portion of inner chamber 130 below dentin sample190. This increased fluid pressure initiates fluid flow in (across orthrough) the dentin tubule orifices in dentin sample 190. Fluid flowcontinues through flow outlet channel 160 of lid 150.

Without being limited by any of the enumerated theories, it is believedthat the limitations of the Pashley cell are addressed by flow cell 100of the present invention as follows. The stacking error found in thePashley flow cell due to the excessive number of “O”-rings (four) andspacers (two) is eliminated by, in some embodiments, requiring no morethan two washers in the flow cell 100. Also, by requiring grooves 118and 158, flow cell 100 reduces, substantially eliminates or eliminatesleaks around the dentin sample caused by sliding or inaccurate placementof the “O”-rings when assembling the Pashley cell.

Next, the washers used in flow cell 100 of the present invention have atleast one flat side for contacting a dentin sample, optionally thewashers have square cross-sections or at least one pair of flat sides,one flat side opposite (or substantially opposite) the other flat sideof the pair, such that one flat side of the pair contacts the lid 150and/or the base and the other flat side of the pair is positioned tocontact a dentin sample for securing the dentin sample within the flowcell, whereas the “O”-rings used with the Pashley flow cell have roundcross-sections. The oppositely situated flat side pairs of the washersof the present invention contact the dentin sample over a consistentarea of the dentin sample, minimizing possible leaks in the system. Suchwashers also eliminate the inability to consistently determine the areaof the dentin sample exposed to the fluid in the flow cell caused byvarying width of the area of the sample/“O” ring contact line due to theflattening out of the round cross-section of the Pashley type “O”-ringswhen pressure is exerted during sealing of the Pashley cell. As areminder, the inability to consistently determine the area of the dentinsample exposed to the fluid in the Pashley flow cell may result in aninconsistent measurement of permeability through the dentin when usingthe Pashley flow cell.

Another issue with the Pashley flow cell relates to the assembly of thePashley flow cell, lid component 50 is screwed onto base component 10.The rotational movement of the lid component 50 with respect to the basecomponent 10 often causes rotation of the dentin sample, the “O”-ringsand the spacers, which can result in leaks around the dentin sample. Inflow cell 100, leaks around dentin sample 190 are minimized by fasteninglid 150 onto bottom component 110 using at least one press seal (i.e., aseal accomplished without rotational movement of the lid 150 relative tothe bottom component 110) fastener 186.

Yet another issue with the Pashley flow cell relates to its “press-fit”connections from inlet and outlet tubes to the inlet 22 and outlet 24channels. The “press-fit” connections often leak, resulting ininaccuracies in flow rate measurements. In certain embodiments, at leastone of inlet channel 144; optional secondary flow inlet channel 142;venting channel 148 and optional secondary venting channel 146 used inflow cell 100 of the present invention have “threaded” connections toinlet and outlet tubes. Specifically, in some embodiments, at least oneof inlet channel 144; optional secondary inlet channel 142; ventingchannel 148; and optional secondary venting channel 146 are machined soas to have “threaded” connections to inlet and outlet tubes via femaleor otherwise compatible treaded tube ends or adapters, such as thoseavailable from Upchurch—IDEX health and Science, Bristol, Conn. orSwagelok, Solon Ohio, and may be composed of metals such as stainlesssteel, polymers, or other non-reactive materials.

The Pashley method further fails to address the issue of air bubbleswhich tend to aggregate under the dentin samples in flow cells duringdentin permeability measurements. Again, without being limited by theoryin any of the following, it is believed that removal (or reduction) ofair bubbles from below dentin sample 190 in inventive flow cell 100 isaccomplished by positioning (e.g., by rotating or tilting) the cell suchthat the angled (by the angle θ) venting channel 148 forms a negativeangle φ relative to a top side of a horizontal cross-sectional planeX″Y″ through the flow cell 100 due to the positioning, the horizontalcross-sectional plane X″Y″ intersecting the inner end 148 a of ventingchannel 148 and angle φ having its vertex at the point of intersectionof the inner end 148 a and the horizontal cross-sectional plane X″Y″.Angle φ, as measured from the top side of the horizontal cross-sectionalplane X″Y″ clockwise (as illustrated at FIG. 10), ranges from greaterthan about 0°, optionally from about 15° to about 85°, optionally fromabout 25° to about 55°, or optionally from about 30° to about 45°. Asillustrated in FIG. 10, rotating or tilting the flow cell 100 clockwise(as illustrated by directional arrow “r”) until venting channel 148forms the described angle φ of greater than about 0° permits removal ofair in the form of air bubble(s) 136. The lower density (relative to thefluid) air bubbles 136 (with directional arrows) flow in a vertical (orsubstantially vertical) direction (i.e., moving positively with respectto the illustrated z″ axis) out from inner chamber 130, through ventingchannel 148 and optional secondary venting channel 146, and, then, outof the flow cell 100. (For purposes of illustrating angle φ, FIG. 10shows the cross-sectional plane X″Y″ perpendicular to, and coming out ofthe paper along the axis x″.)

FIG. 11 is a schematic flow chart drawing, explaining the equipmentlay-out for use in the method of measuring the permeability of dentinaccording to the current invention. The figure shows flow cell 100, as a“black box” in the schematic drawing. Though this is one possiblelay-out of for the equipment, it is to be understood that other possiblelay-outs would also be useful in the method of measuring thepermeability of dentin according to the current invention.

The schematic flow chart drawing includes, in flow communication:pressure generating tank 220; fluid source 230; flow meter 242; pressureregulator 224; pressure gauges 226 and 248; tubes 222, 234, 238, and254; and valves 228, 236, 246, and 256. Tube 222 connects pressuregenerating tank 220 to fluid source 230. A fluid source 230 is providedhaving a vessel (or container) with a cross-sectional area large enoughto prevent a detectable change in fluid level height from loss of fluidto the system apparatus. For example a liter vessel having across-sectional diameter of 10 cm could be used where the loss of fluidfrom the container during the measurement would about 0.5 ml, The vessel(or container) of fluid source 230 is filled with sufficient fluid 232to define a fluid level plane perpendicular to the vessel (or container)wall. The fluid source 230 is positioned at a height Δh (i.e., thedistance from the top of fluid level in the fluid source vessel to thetop of the dentin sample in the flow cell 100). In certain embodiments,the Δh is chosen to provide a pressure (as determine by the static fluidpressure formula) comparable to pulpal pressure, namely from about 0.2psi±0.05 psi. The static fluid pressure formula is ρgh, whereρ=m/V=fluid density, g=acceleration of gravity, and h (or, in thepresent case, Δh)=depth of fluid

Fluid source 230 could be plastic, metal or glass. For example, fluidsource 230 could be a one-liter media bottle supplied by Kimble ChaseLife Science and Research Products LLC, Vineland, N.J., with a GL-45Q-type Bottle cap 3way ¼-28 fitting ports (Fisher Scientific #00945Q-3).Fluid 232 may be water, distilled water, or de-ionized water (DI).

Pressurized inert gas flows from pressure generating tank 220 throughvalve 228, pressure regulator 224 and pressure gauge 226, and into theheadspace above fluid 232 in fluid source 230. Tube 234 and valve 236are located on and, as earlier noted, in flow communication with fluidsource 230, and are used for venting fluid source 230, if necessary.

The pressurization of fluid source 230 by pressure generating tank 220acting as a pumping mechanism (or source of pressure) for pumping fluidinto flow cell 100. Other pumping mechanisms (or sources of pressure)include, but are not limited to, static fluid pressure, piston pumps,rotary piston pumps, diaphragm pumps, gear pumps, or double-actionpiston pumps.

The pressurization of fluid source 230 causes fluid 232 to exit fluidsource 230 through tube 238. The fluid in tube 238 passes through flowmeter 242, valve 246, and pressure gauge 248, and enters flow cell 100through flow inlet channel 144 (or, optionally via secondary flow inletchannel 142) (see FIG. 8). Tube 254 is connected to and, as earliernoted, in flow communication with venting channel 148 (or, optionallyvia secondary vent channel 146 (see FIG. 8) of flow cell 100. Valve 256is located on tube 254 to bleed residual air (or, air bubbles 136)located in the portion of inner chamber 130 below dentin sample 190 atthe start of a dentin permeability measurement. Fluid exits flow cell100 via through flow outlet channel 160 of lid 150 as shown by 252 inFIG. 11.

In one embodiment, the pressure generating tank 220 shown in theschematic drawing of FIG. 11 is a pressurized tank capable of providinga pressure in the apparatus, such as by stand alone laboratory tanks orair compressor. In certain embodiments, the pressure generating tank 220provides pressure of up to 2000 psi. Such pressure generating tanks canbe obtained from a number of known suppliers. Purified air could beused, as could be inert gases such as nitrogen or argon. In oneembodiment, pressure generating tank 220 using nitrogen gas, either“high purity” or “ultra high purity” can be obtained from Air Gas,Radnor, Pa. An example of a pressure generating tank suitable for use inthe present invention includes the N2 Cylinder HP300 supplied by AirGas, Radnor, Pa. Optionally, the gas may be supplied from “house lines”external to the testing location, provided the pressure from the “houselines” is sufficient to perform the disclosed permeability test.

Pressure regulator 224 is an adjustable high precision regulator. Asused herein, a “high precision regulator” means a regulator capable ofmaintaining a pressure of less than or equal to 30 psi (or about 30psi), optionally less than or equal to 20 psi (or about 20 psi),optionally less than or equal to 15 psi (or about 15 psi), optionallyless than or equal to 10 psi (or about 10 psi), optionally less than orequal to 5 psi (or about 5 psi), optionally less than or equal to 2.5psi (or about 2.5 psi) and optionally from about 0.001 psi, optionally0.01 psi (or about 0.01 psi), optionally 0.1 psi (or about 0.1 psi),optionally 0.25 psi (or about 0.25 psi), or optionally 0.5 psi (or about0.5 psi), in all cases, without fluctuations, for a period of at least10 minutes, optionally 15 minutes, optionally 30 minutes, or optionally60 minutes. The term “fluctuation (s)” as used herein means measurementvariation(s) of greater than or equal to ±0.1 psi (or about 0.1 psi),optionally greater than or equal to ±0.01 psi (or about ±0.01 psi),optionally greater than or equal to ±0.005 psi (or about 0.005 psi), oroptionally greater than or equal to ±0.001 psi (or about 0.001 psi). Incertain embodiments, the high precision regulator provides a pressure inthe apparatus such that the fluid flow rate in the apparatus ranges from0 (or about 0) to about 200, optionally from about 0 (or about 0) 0 (orabout 0) to about 85, or optionally from about 0 (or about 0) to about20 micro-liter/minute. An example of a high precision regulator suitablefor use in the present invention is the Type-10LR pressure regulatorsupplied by Marsh Bellofram (Newell, W. Va.). Pressure gauges 226 and248 may, in some embodiments, may be precision digital test gauges suchas Types 2089, 2086, and 2084 supplied by Ashcroft (Huntington Beach,Calif.). Optionally, the apparatus of the present invention can employat least two pressure regulators, a first gross pressure regulatorcapable of maintaining a pressure for a given or certain period of timeand a second high precision pressure regulator capable of maintaining apressure of less than or equal to 20 psi (or about 20 psi), optionallyless than or equal to 15 psi (or about 15 psi), optionally less than orequal to 10 psi (or about 10 psi), optionally less than or equal to 5psi (or about 5 psi), or optionally less than or equal to 2.5 psi (orabout 2.5 psi), without fluctuations, for a period of at least 10minutes, optionally 15 minutes, optionally 30 minutes, or optionally 60minutes.

In one embodiment, the flow rate meter 242 is a high precision flowmeter. When used to describe the flow rate meter, the phrase “highprecision” means a flow meter having an instrument resolution of belowabout 0.5 microliter per minute, or optionally below about 0.5nanoliters. The flow meter can be a manual or digital flow meter. Flowmeter 242 acts as a measuring device suitable for measuring and/ordetermining hydraulic conductance through dentin sample 190. In certainembodiments, the flow rate meter is calibrated to measure fluid flowrates of from about 0 to about 200, optionally from about 0 to about 85,or optionally from about 0 to about 20 microliter/minute. Examples ofmanual flow rate meters that can be used include those supplied byGilmont Instruments (Barrington, Ill.), including the direct readingflowmeter Gilmont Flowmeter GF2000 and the correlated flowmeter GilmontFlowmeter GF3000. Examples of digital flow rate meters that can be usedinclude the Sensirion SLG1430-025 flowmeter supplied by The SensirionCo. (Westlake Village, Calif.) and such flow meters supplied byBronkhorst High-Tech (Bethlehem, Pa.) as the thermal liquid massflowmeter Micro-FLOW series L01 Digital Mass Flow Meter. In someembodiments, a second flow rate meter may be used with flow rate meter242 to confirm that the fluid flow rate in the system of the presentinvention falls within the range that flow rate meter 242 is calibratedto measure (as described above). In other embodiments, one flow ratemeter (manual) could be used to verify the more accurate reading of asecond, digital flow rate meter.

Tubes 222, 234, 238, and 254, may be metal or plastic. In oneembodiment, the tubes are Tube Tefzel (Natural 1/16×0.040×50 ft),available from Upchurch—IDEX health and Science, Bristol, Conn. Valves228, 236, 246, and 256 are used to control flow through the testapparatus, or to isolate sections of the test apparatus. The valves mustbe sized to fit with the rest of the test apparatus. In one embodiment,the valves are 2-Way Valve Bio with ⅛ in Fittings, available fromUpchurch—IDEX health and Science, Bristol, Conn.

Because the permeability flow cell and hydraulic conductance system ofthe present invention addresses the inaccuracies and potential sourcesof leakage associated with the Pashley cell, it generally takes lessthan 5 (or about 5) minutes, optionally, less than 4 (or about 4)minutes, optionally, less than 3 (or about 3) minutes, or optionallyless than 2 (or about 2) minutes for the apparatus' system to achievestability of measurement. The phrase “stability of measurement”, as usedherein, means substantially no fluctuation in the fluid flow ratemeasurement readings, namely, no fluctuations in the fluid flow ratemeasurement readings of less than or equal to ±0.010 grams per hour,optionally less than or equal to ±0.005 grams per hour, or optionallyless than or equal to ±0.001 grams per hour.

The present invention will be better understood from a consideration ofthe following illustrative examples.

EXAMPLES

The following examples are illustrative only and should not be construedas limiting the invention in any way. Those skilled in the art willappreciate that variations are possible which are within the spirit andscope of the appended claims.

Example 1

An in vitro study suing prepared dentin samples to evaluate treatmentwith formulations containing varying amounts of potassium oxalate (KO)as shown in Table 1.

Human dentin samples from molar teeth are used in the study. The samplesare cut from the crown resulting in about one to three samples pertooth, each having a diameter of 10.7±0.5 mm and a thickness of0.54+/−0.05 mm. The above cutting process leaves behind a smear layer oneach surface of the dentin sample. The smear layer is removed by etchingeach dentin sample with 6% citric acid for 3 minutes in conjunction withsonication (sonications in this Example 1 and Example 2 were performedusing a SharperTek CD-4800 ultrasonic cleaner supplied by Sharpertek USA[Pontiac, Mich.]). After etching, the dentin samples are again sonicatedas described above in di-H₂O for 1.5 minutes to thoroughly clean thesample. The dentin samples, as described above, exhibit magnificationproperties when viewing through one side of the dentin sample anddemagnification when viewing through the other side. The samples arestored in vials, magnification side facing upward, with a moistenedtowelette (i.e., Kimwipe with di-H₂O) within the capped vial to preventthe dehydration of the samples.

-   -   A. The system layout of FIG. 11 is prepared and primed for use        in the permeation study as follows:        -   A1. The following system units are turned on:            -   pressure generating tank 220;            -   flow meter (digital) 242; and            -   pressure gauges 226 and 248;        -   A2. Vent valve 236 is opened,        -   A3. The dentin sample 190 is placed in flow cell 100,            verifying that magnification side is facing upwards. The            flow cell 100 is made of an optically clear material (i.e.,            clear acrylic),        -   A4. A syringe is filled with di-H2O (DI) and connected to            the flow cell inlet 144, the DI is present in the syringe at            a volume equal to at least two times the volume of the fluid            cell 100,        -   A5. System valve 256 is opened to permit fluid introduced            from the syringe to flow out,        -   A6. Flow cell 100 is rotated about 45°, moving venting            channel 148 upwards and inlet channel 144 downwards; The            syringe is pressed in pulses to provide a fluid flow into            the cell 100,        -   A7. The syringe is pressed in pulses to provide a fluid flow            into the cell 100,        -   A8. Cell is rotated to view the bottom (i.e.,            demagnification side) of the dentin sample to verify the            presence or absence of air bubbles;        -   A9. Steps A7 and A8 are repeated until no air bubbles are            present;        -   A10. Once all air bubbles are removed, system valve 256 is            closed and syringe is removed.        -   A11. Cell 100 is connected via tubing 238 in flow            communication with valve 246 and valve 246 is opened, (steps            A3 to A11 take less than about 1 to 2 minutes). (When using            the flow cell of the present invention, steps A3 to A11            should generally take no more than 5 minutes, optionally            less than 3 minutes, optionally less than 2 minutes,            optionally less than 1 minute.)        -   A12. If the fluid flow (as measured by flow meter 242) is            below about 15 microliter/min, under the head-pressure due            to gravity (from Δh), valve 228 is opened providing pressure            from pressure generating tank 220 for about 5-10 seconds and            then valve 236 is closed,        -   A13. Once valve 236 is closed, the pressure is adjusted via            pressure regulator 224 to establish about 15 microliter/min            fluid flow rate,        -   A14. Once stable 15 microliter/min fluid flow rate is            established, close valve 246,        -   A15. The top surface of dentin sample 190 is dried with            Kimwipe (or pipette) through flow outlet channel 160;            preparing for commencement of Treatment Protocol    -   B. Treatment Protocol: A formulation of Table A is applied to        the dentin sample of step A (above) as follows:        -   B1. 200 microliters of the formulation is applied to dentin            sample 190 using a pipette, and is left on the dentin sample            for about 1 minute,        -   B2. The formulation is, then, removed (or, taken up) from            the dentin sample using a Kimwipe (or pipette), then 200            microliters of deionized (D.I.) water is applied onto dentin            sample 190 using a pipette, and is left on the dentin sample            for about 1 minute,        -   B3. The DI water is, then, removed using a Kimwipe (or            pipette),        -   B4. Steps B1 through B3 are, then, repeated two additional            times.    -   C. The permeation study with treated dentin sample 190 is        performed using the pre-prepared/primed system of part A as        follows (i.e.,):        -   C1. Following steps of part A, system valve 246 is opened to            start fluid flow;        -   C2. The system is permitted to run for about 5 minutes to            reach equilibrium for experimental fluid flow rate reading            from fluid flow meter 242, and        -   C3. System valve 246 is turned off.    -   D. Treatment Protocol (Steps B1 to B4) and Permeation Study        (Steps C1 to C3) are repeated for each formulation in Table 1        until the earliest of: i) the flow rates of all 15 treatments        are measured (i.e., mimicking about 1 week of use); or ii) no        flow rate is observed through the dentin sample.

The data is normalized by calculating the residual permeability (RP).The RP of dentin sample after a treatment is calculated as follows:

${RP}_{x} = \frac{{Flow}\mspace{14mu} {Rate}_{x}}{{Flow}\mspace{14mu} {Rate}_{0}}$

where: Flow Rate_(x) is the measured flow rate after each of xtreatments (0 to n), where n is the total number of treatments, and FlowRate₀ is the measured flow rate prior to any treatment.

Though typically presented in terms of RP, this step may be omitted whenusing the apparatus (including flow cell) and methods of the presentinvention in view of present invention's ability to standardize flowrates (e.g, at 15 microliters/min) across each dentin sample.

The formulations of Table 1 were prepared using conventional mixingtechnology.

TABLE 1 Dentin Sample Treatment Formulations Formulations based onPotassium Oxalate (KO) Content 0.5% KO 1.0% KO 1.5% KO 2.0% KO 0.0a% KO0.0b% KO Concentration Concentration Concentration ConcentrationConcentration Concentration Ingredient (%) (%) (%) (%) (%) (%) 190 proofethyl 22.6530 22.6530 22.6530 22.6530 22.6530 22.6530 alcohol Menthol0.0323 0.0323 0.0323 0.0323 0.0323 0.0323 Thymol 0.0639 0.0639 0.06390.0639 0.0639 0.0639 Methyl Salicylate 0.0660 0.0660 0.0660 0.06600.0660 0.0660 Eucalyptol 0.0922 0.0922 0.0922 0.0922 0.0922 0.0922Flavor 0.0850 0.0850 0.0850 0.0850 0.0850 0.0850 Poloxamer 407 0.25000.2500 0.2500 0.2500 0.2500 0.2500 Sorbitol Solution, 20.0000 20.000020.0000 20.0000 20.0000 20.0000 70% Sucralose NF 0.0300 0.0500 0.03000.0300 0.0300 0.0300 Potassium Oxalate 0.5000 1.0000 1.5000 2.0000 — —Sodium Fluoride 0.0221 0.0221 0.0221 0.0221 0.0221 — Dye 0.0005 0.00050.0005 0.0005 0.0005 0.0005 Deionized Water QS to 100 QS to 100 QS to100 QS to 100 QS to 100 QS to 100 pH 3.5 3.5 3.5 3.5 3.5 3.5

Table 2 shows the formulations used in the study, as well as theresidual permeability after every three treatments (with StandardDeviation [SD] of measurement). Formulations labeled “0.0a” and “0.0b”were controls.

TABLE 2 Residual Permeability of Potassium Oxalate treated dentinsamples. Number of Treatments (Residual Permeability ± SD) % KO 0 3 6 912 15 0.5 1.000 ± 0.000 0.872 ± 0.127 0.832 ± 0.142 0.723 ± 0.194 0.603± 0.241 0.467 ± 0.255 1.0 1.000 ± 0.000 0.814 ± 0.106 0.542 ± 0.2230.304 ± 0.198 0.152 ± 0.123 0.073 ± 0.055 1.5 1.000 ± 0.000 0.506 ±0.075 0.113 ± 0.022 0.025 ± 0.004 0.018 ± 0.002 0.017 ± 0.004 2.0 1.000± 0.000 0.336 ± 0.044 0.050 ± 0.007 0.022 ± 0.005 0.019 ± 0.002 0.019 ±0.002 0.0a 1.000 ± 0.000 0.997 ± 0.003 0.990 ± 0.007 0.973 ± 0.021 0.966± 0.029 0.955 ± 0.045 0.0b 1.000 ± 0.000 1.032 ± 0.006 1.041 ± 0.0171.054 ± 0.016 1.056 ± 0.015 1.060 ± 0.017 SD = Standard Deviation

The table shows that as the number of treatments increases, the residualpermeability (or, permeability of the dentin samples) decreases for allKO containing formulations. In addition, as the KO percent in thetreatment formulation increases, the rate of decrease of the residualpermeability increases. The reduction of residual permeability of dentinafter treatments corresponds to occlusion efficacy of treatmenttechnology. The residual permeability may be plotted as a function oftreatments and compared against control/other formulations. The resultsfurther indicate that the high throughput apparatus and methods of thepresent invention do not compromise integrity of the generated data.

Example 2

The versatility of the high throughput apparatus (including the flowcell) and methods of the present invention is further illustrated by itsuse in the treatment “challenge” procedure described below. The timeperiod from the removal of the dentin sample from the inventive flowcell to obtaining reliable flow rate data (after reincorporation of thedentin sample into the inventive flow cell and apparatus according tothe procedure outlined below) can be less than 5 minutes, optionally 3minutes, optionally 2 minutes, or optionally 1 minute. The reliabilityof the flow rate data results from the dependability, reliability andpredictability of the setup and the performance of the apparatus(including the flow cell) of the present invention, coupled with therecognition that data integrity is not compromised.

Treatment Challenge Procedure

The treatment technologies applied to dentin samples (e.g., theformulations of Table 1) can be challenged (i.e., brushing, acid,sonication etc., alone or in combination) and, then, evaluated using theflow cell, apparatus and methods of the present invention using thefollowing procedure:

-   -   E. Performing step A1 through D of Example 1, followed by:        -   E1. The top of flow cell 100 is removed from the bottom            component of flow cell,        -   E2. The washer location on the dentin sample is marked,            (this step can also be performed after step A3 in Example            1).        -   E3. The dentin sample is removed from the flow cell 100,        -   E4. The dentin sample is challenged by placing dentin sample            into a vial of hydrodroxylapatite saturated lactic acid            (˜pH=5.0) and sonicated for about 90 seconds,        -   E5. The dentin sample is rinsed in a pool of DI,        -   E6. Steps A3 through A11 of Example 1 are performed to prime            (i.e., remove air bubbles) the flow cell 100 and reestablish            conductivity of apparatus system,        -   E7. The fluid flow rate is then obtained from flow rate            meter 242.            The time period from the removal of the dentin sample from            the flow cell to obtaining flow rate measurement data takes            less than 2 minutes and, in some cases, less than 1 minute.

What is claimed is:
 1. An apparatus for measuring hydraulic conductance of dentin samples, comprising; e. a flow cell comprising; i. a flow inlet channel having an inner opening, the flow inlet extending from the inner opening outwardly from the flow cell; ii. a flow outlet channel in flow communication with the flow inlet channel; and iii. at least one dentin sample securing mechanism positioned to secure a dentin sample between the flow inlet channel and the flow outlet channel; f. a pumping mechanism for exerting a pressure to pump a fluid into the flow cell through the flow inlet channel, through a dentin sample and out of the flow cell through the flow outlet channel; g. a pressure regulator in flow communication with the pumping mechanism for regulating the pressure exerted by the pumping mechanism; and h. at least one flow meter in measuring contact with a fluid pumped by the pumping mechanism for directly measuring the flow rate of a fluid pumped into the flow cell and through the dentin sample.
 2. The apparatus according to claim 1 wherein the pressure regulator is an adjustable pressure regulator capable of maintaining a pressure in the apparatus of less than or equal to 30 psi without fluctuations in the maintained pressure of greater than or equal to ±about 0.1 psi for a period of at least 10 minutes.
 3. The apparatus according to claim 2 wherein the pressure regulator is capable of maintaining a pressure in the apparatus of from about 0.001 psi to about 5 psi without fluctuations in the maintained pressure of greater than or equal to ±about 0.1 psi for a period of at least 10 minutes.
 4. The apparatus according to claim 1 wherein the pressure regulator is capable of maintaining a pressure in the apparatus of less than or equal to 20 psi without fluctuations in the maintained pressure of greater than or equal to ±about 0.01 psi for a period of at least 10 minutes.
 5. The apparatus according to claim 4 wherein the pressure regulator is capable of maintaining a pressure in the apparatus of less than or equal to 20 psi without fluctuations in the maintained pressure of greater than or equal to ±about 0.001 psi for a period of at least 10 minutes.
 6. The apparatus according to claim 1 wherein the pressure regulator is an adjustable pressure regulator capable of maintaining a pressure in the apparatus of less than or equal to 20 psi without fluctuations in the maintained pressure of greater than or equal to ±about 0.1 psi for a period of at least 20 minutes.
 7. The apparatus according to claim 1 comprising a first and second flow meter, the first flow meter for directly measuring the flow rate of a fluid pumped into the flow cell and through the dentin sample and calibrated to measure fluid flow rates of from about 0 microliters per minute to about 200 microliters per minute, the second flow meter for confirming that the flow rate of a fluid pumped into the flow cell and through the dentin sample falls within the flow rate range of from about 0.5 microliters per minute to about 85 microliters per minute.
 8. An apparatus for measuring hydraulic conductance of dentin samples, comprising a fluid flow rate standardization mechanism, comprising: iv. a pumping mechanism for pumping a fluid through the apparatus; v. at least one adjustable high precision flow regulator for maintaining a pressure in the apparatus of less than or equal to 5 psi without fluctuations in the maintained pressure of greater than or equal to ±about 0.1 psi for a period of at least 10 minutes; and vi. at least one fluid flow meter for measuring the fluid flow rate across a dentin sample, wherein the fluid flow rate is standardized across different dentin samples for establishing a single fluid flow rate as the control against which the flow rate of the different dentin samples, after modification of the dentin samples, is compared.
 9. A method for measuring the hydraulic conductance through a dentin sample, comprising the steps of: g. providing a pumping mechanism for pumping a fluid through an apparatus; h. pumping a fluid through the apparatus i. providing at least one adjustable high precision flow regulator for maintaining a pressure in the apparatus of less than or equal to 20 psi without fluctuations in the maintained pressure, for a period of at least 10 minutes, of greater than or equal to ±about 0.1 psi; j. at least one fluid flow meter for measuring the fluid flow rate across a dentin sample; k. directing the fluid flow through a dentin sample; and l. noting the fluid flow rate through the dentin sample indicated by the fluid flow meter wherein steps a. through f. are repeated for at least one other dentin sample and further wherein the flow regulator is adjusted such that the flow rate across the at least one other dentin sample(s) equals the flow rate of the first dentin sample. 